Direct PCR from E. coli colonies

John Nash nash at
Tue Jul 11 15:17:07 EST 1995

In article <DBJxqL.Azr at>,
William D. Woolfolk <wdw7m at> wrote:

>Yes, you can use a similar method.  I have frequently amplified directly 
>from bacteria by pulling up a small amount of bacterial material with a 
>pipet tip, and adding it to a 50ul PCR reaction with the Taq omitted.  I 
>heat to 95C for 15 minutes,then add Taq and cycle as I would for normal 
>DNA template-based PCR.  The only problem I ever had was using a massive 
>clump of bacteria, which decreased amplification some.  A small swipe is 
>more than adequate for PCR, so do not overdo it.  Good luck.

A 10 ul reaction works fine, and (in my hands) one doesn't need to
heat-treat for 15 min (but I do use a 2 min 95 deg first step after
adding Taq).  Just touching a micropipette tip to the colony (you
don't need to see the cells you've removed) and mixing it in with the
master mix also suffices to give nice bands.

John Nash                       Institute for Biological Sciences      *
Email: nash at   National Research Council of Canada  * .*
World Wide Web:            *
         Disclaimer: Opinions are mine, not NRC's

More information about the Methods mailing list